Project Summary/Abstract Exosome research has grown exponentially due to the recognition of the potential roles of exosomes in pathophysiological processes, including cancer. However, due to technical challenges, isolated nanovesicles constitute a heterogeneous population and this has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions in vivo, and has limited their translational application. The state-of- the-art technology, asymmetric-flow field-flow fractionation (AF4), exhibits unique capability to separate nanoparticles and has been widely utilized to characterize nanoparticles and polymers in the pharmaceutical industry as well as various biological macromolecules, protein complexes and viruses. The objective of this study is to develop and validate the application of AF4 in exosome isolation and identification of novel nanovesicles using pancreatic cancer as a model system. We will evaluate its application in analyzing exosomes isolated from a panel of established human pancreatic cancer cell lines (Aim 1). We will further develop and optimize the AF4 methodology to apply it to complex biological specimens such as blood plasma from human subjects (Aim 2). Lastly, we will validate the AF4 application for the fractionation and characterization of distinct exosome subpopulations and identification of other novel nanovesicles using specimens (blood plasma and tumor tissues) isolated from pancreatic patients with newly diagnosed disease and at different stages of disease as well as patients undergoing treatment (Aim 3). We predict that AF4 in combination with sensitive molecular assays can serve as an improved analytical tool for the isolation of specific nanovesicle subpopulations, thereby addressing the complexities of vesicle heterogeneity.